Known and typical prokaryotic expression plasmids contain, in addition to one or several antibiotic resistance gene(s), additional DNA sequences which are not required and burden the cell metabolism. These include DNA sequences resulting from the cloning process, DNA segments that are relics from multipurpose vectors such as specific promoters for the in vitro synthesis of mRNA and specific phage replication origins for the synthesis of single stranded DNA and extend to rudimentary duplicated vector sequences which can be the cause of undesired plasmid rearrangements.
The presence of a plasmid and in particular of an expression vector is an additional metabolic burden for the cell. This results in a selection pressure which favours the formation of cells without plasmids. One method of selecting for cells containing plasmids is antibiotic selection.
Antibiotics such as ampicillin , tetracycline, kanamycin and chloramphenicol are usually used for selection. The use of .beta.-lactam antibiotics such as ampicillin is especially problematic in the production (fermentation) of therapeutic products.
For the reasons discussed above antibiotic plasmid selection, especially by means of .beta.-lactam antibiotics, is not favored in recent methods. In some cases a host/vector system has been developed that proved to be so stable that it was possible to omit a plasmid selection during the preculture and during the main fermentation (Weir, A. N. C. and Mountain, A., EP 0651803). However, as a rule this is associated with a reduced product yield. In cases in which antibiotic selection is indispensable, tetracycline is often used as an alternative to ampicillin (Carter, P. et al., 1992). In contrast to the .beta.-lactam antibiotics, the tetracyclines are not reactive chemical compounds and cannot be inactivated by enzymatic modification during fermentation. The tetracycline resistance gene codes for a protein which modifies the bacterial membrane and thus prevents the antibiotic from entering the cell.
Selection systems have been developed that do not use antibiotic resistance, instead exploiting complementation technology. Struhl, K. and coworkers (Struhl, K. et al., 1976) using imidazole glycerol phosphate dehydratase (HIS3) as an example, show that an appropriate E. coli mutant (hisB) can be directly complemented by means of plasmids containing yeast DNA and that the yeast enzyme coded by HIS3 is functionally expressed in E. coli. This ability of genes to complement mutations in E. coli was used as a selection criterion to clone for example (complementation cloning) other yeast genes (LEU2, URA3, TRP5 and ARG4).
E. coli host strains with a stable mutation (reversion rate &gt;10.sup.-10 ; preferably non-revertable deletion mutants) are required for selection by complementation. However, the reversion rate of known mutations is of the order of magnitude of &lt;10.sup.-10 (Schlegel, H. G., 1981).
The known E. coli laboratory strains differ with regard to their genotype in individual mutations which in many cases were produced by undirected mutagenesis by radiation (X-ray or UV radiation), chemical mutagens (e.g. base analogues, nitrous acid and alkylating compounds) or biological techniques (e.g. phage Mu and transposon mutagenesis (Bachman, B. J., 1986).